Neuroprotective effects of macrostemonoside T on glutamate-induced injury in HT22 cells

Glutamate is a critical excitatory neurotransmitter involved in numerous cellular functions. However, excessive glutamate release can lead to neuronal cell death through oxidative stress, which is implicated in the pathogenesis of various neurological disorders. Therefore, strategies aimed at preventing oxidative stress have emerged as promising therapeutic approaches. Macrostemonoside T (MST), a novel steroidal saponin isolated from the traditional Chinese medicine Allii Macrostemon Bulbus, has demonstrated significant antioxidant activity in previous studies. Nevertheless, its neuroprotective effects against oxidative damage and the underlying molecular mechanisms have not yet been fully elucidated. In this study, we established a glutamate-induced cell injury model using mouse hippocampal neurons (HT22) to investigate the neuroprotective effects of MST and explore its potential mechanisms. A variety of techniques, including DCFH-DA staining, JC-1 staining, Hoechst 33,258 staining, flow cytometry, immunofluorescence staining, ELISA, Western blot analysis, and molecular docking, were employed. The results demonstrated that MST treatment significantly improved the survival of HT22 cells exposed to glutamate. Moreover, MST treatment markedly reduced intracellular levels of reactive oxygen species (ROS) and malondialdehyde while enhancing the activity of antioxidant enzymes such as superoxide dismutase, catalase, and glutathione peroxidase. MST also mitigated mitochondrial dysfunction by inhibiting mitochondrial fission and preserving mitochondrial membrane potential. Additionally, MST reduced excessive autophagy by decreasing autophagy markers and inhibiting the transition from LC3I to LC3II. Furthermore, MST decreased apoptosis rates, lowered pro-apoptotic protein BAX levels, increased the expression of the anti-apoptotic protein Bcl-2, and inhibited the release of apoptosis-inducing factors from mitochondria. Molecular docking analysis showed that MST enhanced PKA activity by blocking endogenous inhibition of PKA, which in turn activated the PKA/CREB/BDNF signalling pathway. Subsequent validation using immunofluorescence and Western blotting further confirmed that MST treatment significantly reversed the glutamate-induced reduction of PRKACA, CREB, p-CREB, and BDNF protein levels. In conclusion, MST is a potent neuroprotective agent that ameliorates glutamate-induced neuronal damage by inhibiting oxidative stress, alleviating mitochondrial dysfunction, reducing autophagy and apoptosis, and activating the PKA/CREB/BDNF signaling pathway.